Abstract The overall goal of the collaborative project is to develop predictive computational models for antibody affinity maturation. This component (Project 3) will contribute the molecular characterization of antibodies and antigens that will be needed to calibrate and test the computational predictions. The experimental work proposed here is based on B-cell lineage analysis, which enables a reconstruction of the history of mutations at the Ig-encoding locus during the trajectory from a naive response to an evolved antibody. Structural and biophysical characterization of the progenitor antibody of a lineage and of its clonal descendants, including the intermediates in the genealogy, provide information about both the output from any stage in the evolution from progenitor to mature antibody and the input to any subsequent stage. These data are essential for any realistic model of affinity maturation and for relating cellular and molecular interactions in a germinal center to their functional consequences. We will produce recombinant influenza virus hemagglutinin (HA) trimeric ectodomain and anthrax toxin protective antigen (PA) as immunogens, analyze the results of those immunizations produced in Project 2, and generate recombinant Fab fragments (chosen because of the characteristics of the lineage from which they derive) for structural and biophysical studies.